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Gravity Signaling


									Gravity Signaling

    Dr. Stanley Roux
     Thomas Bushart
  Bio Labs room 6 & 9
• A constant, (mostly) inescapable, and directional
• Consistency and directionality make it useful an
  information source in addition to, or in lack of,
  other cues
   –   Orientation
   –   Gravitaxis (geotaxis)
   –   Differentiation
   –   Differential growth
  Sensing & Response: Animals
• Specialized cells to sense the movements of fluids
  (endolymph) and solids (otoliths, statocyst)
• Movement/pressure translated by nervous system
  into signals for balance and orientation
End Results: Responses of Plants
       and Single Cells
• Arabidopsis – a multicellular weed
   – Shoot (-) and root (+) growth
• Ceratopteris – an aquatic fern
   – Asymmetric cell division and downward rhizoid growth
• Euglena – a photosynthetic flagellate
   – (-) gravitaxis
• Chara – a non-motile, multicellular algae
   – Cytoplasmic streaming in internodal cells
       Reasoning Backwards
• Root curving
• Asymmetric cell divisions
• Directional movement of a single cell
• Asymmetric cytoplasmic streaming
Growth, cell divisions, movement, and
  streaming all require signaling, therefore
  polarized responses would require polarized
         The Great Unknown
How is the directional force of gravity sensed
  by a single cell? (PE & KE         response)
• Statolith Model
  – The internal downward movement of a dense
• Pressure Model
  – The “sagging” of the cell due to buoyancy
        Starch-Statolith Theory
Conceived from examining the cells required for
  gravity response
• Root columella cells are required for gravity
  perception in roots
   – Ablate columella cells and root will still grow
   – “Gravistimulate” the root (turn the plant on its side) and
     root curvature is impared
• Endodermal cells in shoots are similarly necessary
• Since not the responding cells, likely to be the
  sensing cells
            Columella Cells
• This special root cell type contains large,
  dense, membrane-bound starch grains called
  amyloplasts (present in shoot cells as well)
• Reorientation of the root can result in
  movement of these amyloplasts in the new
  downward direction                  Chen et al. 120(2):343 Plant Phys
      Further Implications for
Mutants with reduced or absent starch
  production would have smaller or absent
• Less starch = less response to gravity
• Microgravity experiments on Arabidopsis
  root curvature
  – WT and starch mutants similar in micro g
  – 1-g centrifuge: WT reorient, starch mutants are
           Pressure Theory
Mass of the protoplast as a whole is
  affected by the gravity vector
• Chara internodal cells and Euglena
  – Lack amyloplasts
  – Manipulations of environment can
    affect cytoplasmic streaming
    response and directional movment
           Pressure and Density
An elastic object more dense than its surroundings
 will sag such that there is compression forces on
 the bottom and tensile forces on the top
   – A balloon filled with water will flatten out when placed
     on a table (water > air)
   – Place the water filled balloon in water and it will take
     the natural shape of the balloon (water = water)
   – Anything inside the water filled balloon will behave
     independently of the media surrounding the balloon
     (i.e. a marble would still sink to the bottom)
• Cells are like a balloon, organelles like marbles
        Change Environment
Cytoplasm is naturally more dense than water
  (and of course air)
• Density of media = density of cytoplasm
  – Euglena remain motile, but loose directionality
  – Chara streaming will become equal
• Density of media > density of cytoplasm
  – Euglena swim downward
  – Chara stream in the opposite direction
  Further Evidence for Pressure
• Hydrostatic pressures applied externally to
  ends of horizontal Chara cells effects
  streaming rates
  – Positive pressures = streaming away from
    stimulated end
  – Negative pressures = streaming towards
    stimulated end
• A vertically oriented cell would experience
  different pressures on each end
 Problems: Ambiguous Evidence
        & Universality
• Some results could lend support to either theory
  depending on how you look at it
   – Starch mutants still have some ability to respond to
     gravity (especially under high g forces)
   – Laser ablation of columella cells doesn’t completely
     remove gravity response
   – Actual movement of a statolith may not be required
   – Germinating rice placed in high density media still
     show some root curvature
• Results from one system may not be applicable to
   – Not all gravity responding cells contain statoliths
   – Chara cells are MUCH larger than most cells (on the
     order of several centimeters long)
            Common Ground
• The force of gravity acts upon some
  element of the cell (statoliths or protoplast)
• The receptor element then acts upon
  something else within the cell to initiate a
  signal for asymmetric signaling
  – In both theories this initial signaling event is
    likely associated with membranes, either
    directly or though cytoskeletal connections
        Necessary Components
•   Cytoskeleton
•   Membranes
•   Proteins
•   Calcium
  Cytoskeleton and Membranes
• Amyloplasts and other organelles are
  enmeshed within an actin network
• Amyloplasts may be restrained by ER
  elements or in contact with specialized ER
• Tension/Compression forces would act
  directly upon plasma membranes
• The Altered Response to Gravity (ARG1)
  gene encodes a putative Dna-J like protein
    Chara Extracellular Matrix
• Degradation of certain cell wall elements
  abolishes gravity response
• RGDS peptides applied to the top end of
  cells inhibit gravity response (tension)
  – RGDS implies integrins
  – Integrins would involve membranes, ECM, and
        Protein Requirement
• The Euglena photoreceptor is inhibited by
  UV-B light which in turn inhibits
  phototaxis. UV-B light can also impair
• Treating the ends of Chara cells with
  proteases or UV irradiation abolishes
• A mutation in the Arabidopsis ARG1
  protein alters gravity response
Implicated in all model systems
• Euglena - Gd, vanadate, and a calcium ionophore
  all inhibit gravitaxis
• Chara – external Ca, nifedipine, La effect
• Arabidopsis – Gd/La and calmodulin & Ca-
  ATPase blockers inhibit gravity reponse, Ca
  gradient seen across gravistimulated root
• IP3 signaling as well?
Hypothetical Signaling Story
              1. Gravity acts upon protoplasm
              2. Force acts through integrins
              3. Ion channels open in a polar
              4. Subsequent responses

              1. Gravity acts on statolith
              2. Statolith in contact with ER
                 activates ion channels, or #2
              3. Responses
 Ceratopteris Spores as a Model
• Single cell both senses and responds to
• Only slightly larger than a typical
  multicellular plant cell
• Earliest detectable response is a gravity
  directed calcium flux
• Known time frame for gravity directed
  polarity fixation
• Subsequent steps are visually apparent
Spore Germination Is Induced by Light and
Polarity Development Is Directed by Gravity

        Period of
                        Downward nuclear    Rhizoid emergence
     gravity fixation
                         migration (24 h)          (72h)
        ( 4-18 h)
                       Calcium Movement


                                                                           Calcium top
                      2500                                                 Calcium Bottom
Flux (fmol/cm2/sec)

                                                                           Calcium side
                                                                           Proton Top
                      1500                                                 Proton side



                              0        5     10     15      20        25

                                  Hour After Germination Initiation
ATP        ADP + Pi
                            Use calcium as the starting point
                               Calcium channels
           “Late” gravity
            responses          Calcium pumps
                               Calcium transmitters

      Ca2+ ions
• Gravitational forces lead to a variety of polar
   – Movement, growth, differentiation
• Mediated by cytoskeletal, membrane, and protein
   – Actin, microtubules, PM & ER, intergrins, ion channels
     and pumps
• Actual receptor(s) and subsequent signaling
  pathway(s) still being investigated
• Chen R., Rosen, E., Masson P. “Gravitropism in
  Higher Plants.” Plant Physiology. (1999) 120:
• Staves, M. “Cytoplasmic streaming and gravity
  sensing in Chara internodal cells.” Planta (1997)
  203: S79-S84
• Chatterjee, A., Porterfield D., Smith, P., Roux, S.
  “Gravity-directed clacium current in germinating
  spores of Ceratopteris richardii.” Planta (2000)
  210: 607-610

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